Despite the imminent threat and devastating ocular injuries by exposure to vesicating agents sulfur mustard (SM), nitrogen mustard (NM) and arsenical vesicant, lewisite (LEW), effective therapies in case of a mass casualty remain elusive. This is mainly due to the lack of established animal injury models and inadequate study on the pathogenesis of eye lesions and associated mechanism/s. An additional practical limitation for such studies has also been a lack of availability of vesicating agents and appropriate facilities to use them in experimental settings. Accordingly, the first and foremost goal of this application is to develop a comprehensive strategy related to establishing vesicant agents (NM, SM and LEW)-induced ocular injury models and define the associated mechanisms. Once achieved, this will help us perform efficacy studies with mechanism-driven therapeutic agents; this is the second major goal of this application. Both our goals are supported by preliminary studies in rabbit corneal organ culture showing that NM exposure increases epithelial thickness, apoptotic cell death, epithelial-stromal separation, levels of VEGF, COX-2 and MMP-9; and that approved prescription agents anti-inflammatory drug dexamethasone and antibiotic doxycycline (an MMP inhibitor) as well as a natural agent silibinin significantly reduce NM-induced epithelial thickness, epithelial-stromal separation, and VEGF, COX-2 and MMP-9 levels, albeit at different levels. Overall, based on above rationale and preliminary data, this application is deliverables driven and proposes to test the hypothesis that most of the mechanisms involved in ocular injuries by vesicating agents NM, SM and LEW follow similar pathways, and that , by targeting those pathways, we would be able to develop effective and broad-spectrum therapies against vesicants-induced ocular injuries. Our specific aims are: 1. Fully characterize and establish ocular injury models with vesicating agents. Specifically, we will assess biological alterations and identify associated mechanisms including those related to inflammation, vesication and neovascularization. 2. Evaluate the efficacy of mechanism-targeted agents (alone or in combination) and silibinin (a pleiotropic agent with strong therapeutic efficacy against blistering agents-induced skin injuries) in treating the ocular injuries by NM. 3. Assess and establish the efficacy of the agent/s found effective in treating NM-induced ocular injuries in Aim 2, for the rescue of SM- and LEW-induced ocular injuries in rabbit. We anticipate that the proposed studies will establish useful ocular injury models with vesicants, and add to our understanding of the mechanism/s of action of NM-, SM- and LEW-induced ocular injuries. In addition, these comprehensive study efforts will identify effective broad spectrum mechanism-based agent/s that alone or in combination provide effective therapies against vesicating agents-induced ocular injuries in humans. PUBLIC HEALTH RELEVANCE: Despite the imminent threat of use as warfare and terrorist agents, effective therapies against vesicating chemical agents, sulfur mustard (SM)-, nitrogen mustard (NM)- and lewisite (LEW)-induced devastating ocular injuries, remain elusive. This is mainly due to the lack of established injury models, and inadequate study on the pathogenesis of eye lesions and associated mechanism/s; inappropriate engineering facilities to use these agents in experimental settings are an additional concern. Accordingly, the goals of this application are: a) develop NM-, SM- and LEW-induced ocular injury models, and define the associated mechanisms, and b) perform efficacy studies with mechanism-driven therapeutic agents. Our goals are supported by preliminary studies in rabbit corneal organ culture showing that NM-causes an increase in the inflammatory, vesication, and angiogenic responses, which are significantly reduced by the treatment with approved anti-inflammatory and antibiotic drugs as well as a natural flavanone, silibinin. Overall, the present application will test the hypothesi that most of the mechanisms involved in ocular injuries by vesicating agents follow similar pathways, and that, by targeting those pathways, we would be able to develop effective and broad-spectrum therapies against vesicants-induced ocular injuries. We anticipate that the study outcomes here will add to our understanding of the mechanism/s of action of NM-, SM- and LEW-induced ocular injuries, and identify effective mechanism-based broad-spectrum agent/s that, alone or in combination, provide safe and effective therapies against vesicating agents-induced ocular injuries in humans.